Initial Screening of Poly(ethylene glycol) Amino Ligands for Affinity Purification of Plasmid DNA in Aqueous Two-Phase Systems.

Gene therapy and DNA vaccination are among the most expected biotechnological and medical advances for the coming years. However, the lack of cost-effective large-scale production and purification of pharmaceutical-grade plasmid DNA (pDNA) still hampers their wide application. Downstream processing, which is mainly chromatography-based, of pDNA remains the key manufacturing step.

Hofmeister effects on protein stability are dependent on the nature of the unfolded state.

The interpretation of a salt's effect on protein stability traditionally discriminates low concentration regimes (<0.3 M), dominated by electrostatic forces, and high concentration regimes, generally described by ion-specific Hofmeister effects. However, increased theoretical and experimental studies have highlighted observations of the Hofmeister phenomena at concentration ranges as low as 0.

Protein folding and quinary interactions: creating cellular organisation through functional disorder.

The marginal stability of globular proteins in the cell is determined by the balance between excluded volume effect and soft interactions. Quinary interactions are a type of soft interactions involved in intracellular organisation and known to have stabilising or destabilising effects on globular proteins. Recent studies suggest that globular proteins have structural flexibility, exhibiting more than one functional state.

Simplified 2,4-dinitrophenylhydrazine spectrophotometric assay for quantification of carbonyls in oxidized proteins.

This work proposes a modification of the 2,4-dinitrophenylhydrazine (DNPH) spectrophotometric assay commonly used to evaluate the concentration of carbonyl groups in oxidized proteins. In this approach NaOH is added to the protein solution after the addition of DNPH, shifting the maximum absorbance wavelength of the derivatized protein from 370 to 450nm. This reduces the interference of DNPH and allows the direct quantification in the sample solution without the need for the precipitation, washing, and resuspension steps that are carried out in the traditional DNPH method.

Influence of secretory leukocyte protease inhibitor-based peptides on elastase activity and their incorporation in hyaluronic acid hydrogels for chronic wound therapy.

Chronic nonhealing skin wounds, such as leg ulcers and pressure sores, represent a major clinical problem and a financial burden for the health care systems. Chronic wounds are characterized by prolonged inflammatory phase that results in high levels of elastase, reactive oxygen species (ROS), and diminished growth factor activity. Under normal physiological conditions, elastase is a powerful host defence and its activity is regulated by endogenous inhibitors.

Characterization of potential elastase inhibitor-peptides regulated by a molecular switch for wound dressings applications.

Elastase plays an important role in wound healing process, degrading damaged tissue and allowing complete tissue recovery. The levels of human neutrophil elastase (HNE) are usually controlled by endogenous inhibitors. However, in the presence of high levels of elastase, like the ones present in chronic wounds, the inhibitors cannot overcome this overproduction and the enzyme starts to degrade the surrounding healthy tissue.

Protein quantification in the presence of poly(ethylene glycol) and dextran using the Bradford method.

Some experimental methodologies require the quantification of protein in the presence of polymers like poly(ethylene glycol) (PEG) and dextran (DEX). In the aqueous two-phase system (ATPS) extraction of biomolecules, the interference of these phase-forming polymers on the Bradford quantification assay is commonly recognized. However, how these polymers interfere has not been reported hitherto.

Preparation of plasmid DNA polyplexes from alkaline lysates by a two-step aqueous two-phase extraction process.

This work studied the possibility of using polyethyleimine (PEI) as an affinity ligand for the purification of plasmid DNA (pDNA) from alkaline lysates using aqueous two-phase systems (ATPSs). The goal was to find conditions under which this cationic polymer could steer the partition of pDNA to the phase where less impurities accumulate. In poly(ethylene glycol) (PEG)/ammonium sulphate systems, neither free nor PEGylated PEI (pPEI) were able to change the partition of pDNA.

Purification of plasmid DNA vectors by aqueous two-phase extraction and hydrophobic interaction chromatography.

The current study explores the possibility of using a polyethyleneglycol(PEG)-ammonium sulphate aqueous two-phase system (ATPS) as an early step in a process for the purification of a model 6.1 kbp plasmid DNA (pDNA) vector. Neutralised alkaline lysates were fed directly to ATPS.

Production of 6-aminopenicillanic acid in aqueous two-phase systems by recombinant Escherichia coli with intracellular penicillin acylase.

Bioconversion of penicillin G in PEG 20000/dextran T 70 aqueous two-phase systems was achieved using the recombinant Escherichia coli A56 (ppA22) with an intracellular penicillin acylase as catalyst. The best conversion conditions were attained for: 7% (w/v) substrate (penicillin G), enzyme activity in bottom phase 52 U ml(-1), pH 7.8, temperature 37 degrees C, reaction time 40 min.